MOLECULAR CLONING, EXPRESSION AND STRUCTURE OF ENAMEL PROTEINS

牙釉质蛋白的分子克隆、表达和结构

• 批准号: 6104743
• 负责人: JOEL ROSENBLOOM
• 金额: $ 11.45万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6104743
• 关键词: RNA splicing; affinity chromatography; amelogenin; animal tissue; circular dichroism; conformation; dental development; dental disorder; fluorescence spectrometry; gene deletion mutation; gene expression; genetic disorder; genetic polymorphism; genetic regulatory element; genetically modified animals; human genetic material tag; human tissue; hydroxyapatites; laboratory mouse; laboratory rabbit; molecular cloning; nucleic acid sequence; point mutation; protein structure function; proteins; sex chromosomes; sex linked trait; site directed mutagenesis; southern blotting; western blottings

The overall goals of this research proposal are to determine the role(s) that individual amelogenin polypeptides play in enamel development, and how alterations in them may result in deranged amelogenesis, such as occurs in the heritable disease, Amelogenesis Imperfecta. With presently available techniques, solubilized amelogenin proteins can be resolved into 10 or more components. Although physiologic processing or artefactual degradation probably contribute to this heterogeneity, recent information from our laboratory suggests that at least some of the heterogeneity arises from the transcription of two divergent genes located on the X and Y chromosomes and from alternative splicing of the primary transcripts. To prove this, we have adopted a combined immunologic and protein analytic approach to isolate and characterize some of the individual components using high resolution chromatographic and electrophoretic techniques. Emphasis will be placed on identifying the translation products of alternatively spliced messages, and the resolved components will be subjected to extensive immunologic and chemical analyses to distinguish them from proteolytically processed components. the cloning and sequence analysis of the normal human amelogenin genes will be completed, and these sequences will be compared to those of genes, amplified by the polymerase chain reaction, in patients with Amelogenesis Imperfecta (AI) in order to better understand the defects involved at the molecular level. In addition, we plan to compare the functional characteristics of the normal and abnormal genes in transgenic mice. Finally, selected amelogenin polypeptides, including normal and mutated sequences that we design as well as peptides corresponding to those found in AI patients, will be expressed by recombinant DNA techniques, in order to evaluate functions of individual peptides, and to formulate and test hypotheses about the roles of amelogenins in enamel development.

该研究建议的总体目标是确定角色 那个单独的氨基蛋白蛋白多肽在搪瓷发育中发挥作用， 它们中的改变如何导致杂乱无章的作用，例如 发生在可遗传的疾病中，症状不完美。 目前 可用的技术，可溶解的氨基蛋白蛋白可以解决 分为10个或更多组件。 虽然生理处理或 人工降解可能有助于这种异质性，最近 我们实验室的信息表明，至少有一些 异质性来自两个不同基因的转录 位于X和Y染色体上，来自 主要成绩单。 为了证明这一点，我们采用了一个合并 免疫学和蛋白质分析方法分离和表征 一些使用高分辨率色谱的单个组件 和电泳技术。 重点将放在识别 剪接消息的翻译产品，以及 已解决的组件将受到广泛的免疫学和 化学分析将它们与蛋白水解处理 成分。 正常人的克隆和序列分析 氨基蛋白蛋白基因将完成，并将这些序列进行比较 对于基因的那些，通过聚合酶链反应放大，在 有障碍的患者（AI）为了更好地理解 分子水平涉及的缺陷。 此外，我们计划 比较正常基因和异常基因的功能特征 在转基因小鼠中。 最后，选定的氨基蛋白蛋白多肽，包括 我们设计的正常和突变序列以及肽 与AI患者中发现的相对应 重组DNA技术，以评估个体的功能 肽，并提出和检验有关有关的作用的假设 搪瓷发育中的氨基蛋白素。